The phosphotyrosine interactome of the insulin receptor family and its substrates IRS-1 and IRS-2

Abstract

The insulin signaling pathway is critical in regulating glucose levels and is associated with diabetes, obesity, and longevity. A tyrosine phosphorylation cascade creates docking sites for protein interactions, initiating subsequent propagation of the signal throughout the cell. The phosphotyrosine interactome of this medically important pathway has not yet been studied comprehensively. We therefore applied quantitative interaction proteomics to exhaustively profile all potential phosphotyrosine-dependent interaction sites in its key players. We targeted and compared insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) as central distributors of the insulin signal, the insulin receptor, the insulin-like growth factor 1 receptor, and the insulin receptor-related receptor. Using the stable isotope labeling by amino acids in cell culture (SILAC) approach with phosphorylated versus non-phosphorylated bait peptides, we found phosphorylation-specific interaction partners for 52 out of 109 investigated sites. In addition, doubly and triply phosphorylated motifs provided insight into the combinatorial effects of phosphorylation events in close proximity to each other. Our results retrieve known interactions and substantially broaden the spectrum of potential interaction partners of IRS-1 and IRS-2. A large number of common interactors rationalize their extensive functional redundancy. However, several proteins involved in signaling and metabolism interact differentially with IRS-1 and IRS-2 and thus provide leads into their different physiological roles. Differences in interactions at the receptor level are reflected in multisite recruitment of SHP2 by the insulin-like growth factor 1 receptor and limited but exclusive interactions with the IRR. In common with other recent reports, our data furthermore hint at non-SH2 or phosphotyrosine-binding domain-mediated phosphotyrosine binding.

Fig. 1.

Proteomic screening for interaction partners of tyrosine phosphorylated sequences. Peptides corresponding to potential tyrosine phosphorylation sites are synthesized in phosphorylated and non-phosphorylated form. Cell populations are metabolically labeled using the SILAC technique to allow discrimination based on different peptide masses. Cell lysate from the population labeled with heavy Arg and Lys is incubated with the phosphorylated version of the peptide, whereas the control cell lysate is incubated with the non-phosphorylated peptide. Eluted proteins from those parallel pulldown experiments are combined and digested with trypsin. Peptides from unspecific background binders appear as pairs with abundance ratios close to 1:1 in the mass spectra. Phosphorylation-specific binders are identified as such through their high abundance ratio between heavy and light labeling states. In a crossover experiment, the incubation scheme is swapped, resulting in inverted ratios.

Fig. 2.

Typical result of a SILAC peptide pulldown experiment, exemplified by the bait peptide IRS-1 Tyr-0891. A, a peptide derived from the interaction partner Grb2 is about 40 times more abundant in the heavy form than in the light form, indicating that Grb2 binds specifically to IRS-1 phosphorylated on Tyr-0891. B, in a crossover experiment (swapped SILAC labels) the abundance ratio of the same peptide is inverted. C, most other proteins have a 1:1 ratio, indicating that they are unspecific binders to the peptides or the magnetic beads that are bound irrespective of phosphorylation. D, a plot of the protein abundance ratios shows that Grb2 and SHP2 are significant outliers in both the Tyr-0891 pulldown and the crossover experiment. Every dot represents one protein.

Fig. 3.

The phosphotyrosine interactome of IRS-1 and IRS-2. Tyr(P)-specific interaction partners obtained in peptide pulldown experiments are depicted as symbols along the primary structure of IRS-1 and IRS-2. Detailed data for every pulldown experiment is provided in supplemental Table 1.

Fig. 4.

The phosphotyrosine interactome of InsR, IGF1R, and IRR. Tyr(P)-specific interaction partners obtained in peptide pulldown experiments are depicted as symbols along the primary structure of the intracellular regions of the receptors. The cytoplasmic part starts at position 968 for the InsR, 961 for the IGF1R, and 944 for the IRR, according to the Swiss-Prot database. Detailed data for every pulldown experiment is provided in supplemental Table 1.

Fig. 5.

Triple labeling pulldown reveals distinct combinatorial effects of double phosphorylation within the NPEY motifs in the insulin receptor family. A, when phosphorylated at the tyrosine within the NPEY motif, the InsR recruits Shc, as demonstrated by the high ratio between the medium-label state and the unlabeled state. If the tyrosine located seven amino acids further N-terminal also carries a phosphorylation, the interaction with Shc is abolished. B and C, when IGF1R or IRR exhibits this phosphorylation pattern, the interaction between Shc and the receptor is not influenced by the second phosphorylation event (supplemental Table 3).

Fig. 6.

Molecular functions potentially influenced by IRS-1 and IRS-2 through direct binding. The Tyr(P)-dependent interaction partners of IRS-1 and IRS-2 identified in this study are grouped according to their function and subcellular location. Stimulatory and inhibitory effects on the pathways are indicated by [⊕] and [⊖], respectively.